The overall objectives are to develop nuclear magnetic resonance (NMR) techniques and use them in concert with other experimental approaches to elucidate the molecular structure and physiologic functions of selected membrane-targeting proteins involved in phototransduction in vision and other signal transduction processes. During the next five years, we will use nuclear magnetic resonance (NMR), fluorescence, microcalorimetry, spin-label EPR, x-ray crystallography, and computational analysis to delineate the structure, dynamics and mechanisms of a family of neuronal calcium sensor proteins (calcium-myristoyl switches) that serve as membrane- targeting regulators in calcium signaling and are linked to retinal and neurological diseases. Our studies will determine the structural basis of: (1) Ca2+-dependent activation of retinal guanylate cyclase (RetGC) by GCAP1, genetically linked to autosomal dominant cone dystrophy; (2) Ca2+-induced inhibition of photoreceptor cyclic nucleotide gated (CNG) channels by CNG-modulin; and (3) Ca2+- dependent activation of retinal L-type Ca2+ channels (CaV1.4) by calcium binding protein-4 (CaBP4), implicated in congenital stationary night blindness. By continuing our intensive study of retinal calcium sensor proteins and by broadening its scope to encompass neuronal homologs and protein targets, we hope to gain an atomic-level understanding of how calcium sensor proteins operate in signal transduction and disease processes. In particular, we want to understand how covalently attached myristoyl groups work in concert with calcium-binding sites and target proteins to guide this family of proteins to specific membrane-bound targets. The specific aims are 3-fold: (1) Determine atomic-level structures of the GCAP proteins bound to retinal guanylate cyclases (RetGCs) to elucidate the Ca2+-dependent activation mechanism of RetGCs and thus provide a structural basis for understanding mechanisms of visual recovery and retinal degenerative diseases; (2) Determine structures of CNG-modulin bound to CNG channels to provide a structural basis for understanding the mechanism of light-adaptation in cone photoreceptors; (3) Determine atomic-level structures of the retinal calcium sensor protein (CaBP4) bound to the retinal L-type voltage-gated Ca2+ channel (CaV1.4) at the rod synapse to understand the Ca2+-dependent regulatory mechanism of ion channels linked to congenital stationary night blindness.